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I present the perspective that the divalent metalome and the metabolome can be modeled as a network of chelating interactions instead of separate entities. I review progress in understanding the complex cellular environment, in particular recent contributions to modeling metabolite-Mg 2+ interactions. I then demonstrate a simple extension of these strategies based approximately on intracellular Escherichia coli concentrations. This model is composed of four divalent metal cations with a range of cellular concentrations and physical properties (Mg 2+ , Ca 2+ , Mn 2+ , and Zn 2+ ), eight representative metabolites, and interaction constants. I applied this model to predict the speciation of divalent metal cations between free and metabolite-chelated species. This approach reveals potentially beneficial properties, including maintenance of free divalent metal cations at biologically relevant concentrations, buffering of free divalent metal cations, and enrichment of functional metabolite-chelated species. While currently limited by available interaction coefficients, this modeling strategy can be generalized to more complex systems. In summary, biochemists should consider the potential of cellular metabolites to form chelating interactions with divalent metal cations.

Interplay between divalent cations (Mg 2+ and Ca 2+ ) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT 30 and dA 30 , with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg 2+ and Ca 2+ , we find that Mg 2+ binds to the minor grooves and phosphate groups, whereas Ca 2+ binds specifically to the minor groove. Both Mg 2+ and Ca 2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA 30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT 30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn 2+ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg 2+ , as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn 2+ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn 2+ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn 2+ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.

TRAAK and TREK2 are two-pore domain K + (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca 2+ , Mg 2+ , and Zn 2+ ) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu 2+ and Zn 2+ than TREK2. In the presence of Cu 2+ , TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu 2+ . High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu 2+ -bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu 2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu 2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis . We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg 2+ and Sr 2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg 2+ . Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of in vitro studies, most of which have used ≥10 mM free Mg 2+ ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg 2+ ions are much lower, with most Mg 2+ ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg 2+ and to estimate cellular concentrations of metabolite-chelated Mg 2+ species, in the representative prokaryotic and eukaryotic systems Escherichia coli , Saccharomyces cerevisiae , and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg 2+ ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg 2+ complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg 2+ should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb 2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni 2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, R p and S p , indicated that Mg 2+ , Mn 2+ , Fe 2+ , and Co 2+ had the highest S p : R p activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb 2+ , suggesting inner-sphere coordination. The pH-rate profiles showed that Pb 2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent p K a and the p K a of metal-bound water. Combining previous reports and our current results, we propose that Pb 2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

Divalent metal cations are essential for many biological processes; however, accurately modeling divalent metal ions has proved a significant challenge for molecular dynamics force fields. Here we show that the choice of ion model influences the observed dynamics in PsaA, a metal binding protein from Streptococcus pneumoniae . We conduct extensive unbiased simulations and free energy calculations of PsaA bound to its cognate ligand Mn 2+ and inhibitory ligand Zn 2+ using three nonbonded ion models: a 12-6 model, a 12-6-4 model, and a multisite model. The observed coordination geometries and metal binding dynamics are sensitive to the choice of ion model, with the most dramatic differences observed in free energy calculations of ion release. We show that the conformational ensemble of Mn-bound PsaA is more similar to the crystallographic metal bound open state. This work extends the current model of PsaA metal binding and provides a framework for the rationalization of experimentally determined metal binding behavior. Our findings support the use of the 12-6-4 ion model for further simulations of divalent cation binding proteins.

Zinc binding to β-amyloid structure could promote amyloid-β aggregation, as well as reactive oxygen species (ROS) production, as suggested in many experimental and theoretical studies. Therefore, the introduction of multifunctional drugs capable of chelating zinc metal ion and inhibiting Aβ aggregation is a promising strategy in the development of AD treatment. The present study has evaluated the efficacy of a new bifunctional peptide drug using molecular docking and molecular dynamics (MD) simulations. This drug comprises two different domains, an inhibitor domain, obtained from the C-terminal hydrophobic region of Aβ, and a Zn 2+ chelating domain, derived from rapeseed meal, merge with a linker. The multifunctionality of the ligand was evaluated using a comprehensive set of MD simulations spanning up to 3.2 μs including Aβ relaxation, ligand-Zn 2+ bilateral interaction, and, more importantly, ligand-Zn 2+ -Aβ 42 trilateral interactions. Analysis of the results strongly indicated that the bifunctional ligand can chelate zinc metal ion and avoid Aβ aggregation simultaneously. The present study illustrated that the proposed ligand has considerable hydrophobic interactions and hydrogen bonding with monomeric Aβ in the presence of zinc metal ion. Therefore, in light of these considerable interactions and contacts, the α-helical structure of Aβ has been enhanced, while the β-sheet formation is prevented and the α-helix native structure is protected. Furthermore, the analysis of interactions between Aβ and ligand-zinc complex revealed that the zinc metal ion is coordinated to Met13, the ending residue of the ligand and merely one residue in Aβ. The results have proven the previous experimental and theoretical findings in the literature about Aβ interactions with zinc metal ion and also Aβ interactions with the first domain of the proposed ligand. Moreover, the current research has evaluated the chelation using MD simulation and linear interaction energy (LIE) methods, and the result has been satisfactorily verified with previous experimental and theoretical (DFT) studies.

The βγ -crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ -crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ -crystallins bind calcium ions. The βγ -crystallin from the tunicate Ciona intestinalis ( Ci - βγ ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci - βγ is valuable for investigating the evolution of the βγ -crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca 2+ and other divalent cations on the stability and aggregation propensity of Ci - βγ and human γS-crystallin (HγS). Beyond Ca 2+ , Ci - βγ is capable of coordinating Mg 2+ , Sr 2+ , Co 2+ , Mn 2+ , Ni 2+ , and Zn 2+ , although only Sr 2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci - βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn 2+ , Ni 2+ , and Co 2+ induce aggregation by interacting with cysteine residues, whereas Cu 2+ -mediated aggregation proceeds via a different binding site.

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg 2+ free . We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg 2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg 2+ /Ca 2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are considered noncompetitive inhibitors that structurally alter reverse transcriptase (RT) and dramatically decrease catalysis. In this report, biochemical analysis with various divalent cations was used to demonstrate that NNRTIs and divalent cation-dNTP complexes are mutually exclusive, inhibiting each other's binding to RT/primer/template (RT-P/T) complexes. The binding of catalytically competent divalent cation-dNTP complexes to RT-P/T was measured with Mg 2+ , Mn 2+ , Zn 2+ , Co 2+ , and Ni 2+ using Ca 2+ , a noncatalytic cation, for displacement. Binding strength order was Mn 2+ ≈ Zn 2+ ≫ Co 2+ > Mg 2+ ≈ Ni 2+ . Consistent with but not exclusive to mutually exclusive binding, primer extension assays showed that stronger divalent cation-dNTP complexes were more resistant to NNRTIs (efavirenz (EFV), rilpivirine (RPV), and nevirapine (NVP)). Filtration assays demonstrated that divalent cation-dNTP complexes inhibited the binding of 14 C-labeled EFV to RT-P/T with stronger binding complexes formed with Mn 2+ inhibiting more potently than those with Mg 2+ . Conversely, filter binding assays demonstrated that EFV inhibited 3 H-labeled dNTP binding to RT-P/T complexes with displacement of Mn 2+ -dNTP complexes requiring much greater concentrations of EFV than the more weakly bound Mg 2+ -dNTP complexes. EFV bound relatively weakly to the NNRTI resistant K103N RT; but, binding was modestly enhanced in the presence of P/T, and EFV was easily displaced by divalent cation-dNTP complexes. This suggests that K103N overcomes EFV inhibition mostly by binding more weakly to the drug and is in contrast to other reports that indicate K103N has little to no effect on drug or dNTP binding. Overall, this biochemical analysis supports recent biophysical analyses of NNRTI-RT interactions that indicate mutually exclusive binding.

Pb 2+ exposure leads to diverse neurological disorders; however, the mechanism of Pb 2+ -induced neurotoxicity is not clearly understood. Here we demonstrate that Pb 2+ binds to EF-hands in apo-DREAM (downstream regulatory element antagonist modulator) with a lower equilibrium dissociation constant ( K d = 20 ± 2 nM) than Ca 2+ ( K d = 1 μM). Based on the Trp169 emission and CD spectra, we report that Pb 2+ association triggers changes in the protein secondary and tertiary structures that are analogous to those previously observed for Ca 2+ -bound protein. The hydrophobic cavity in the C-terminal domain of DREAM is solvent exposed in the presence of Pb 2+ as determined using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). Pb 2+ association with DREAM also modulates interactions between DREAM and its intracellular partners as evident from the fact that Pb 2+ -bound DREAM associates with peptide-based model systems, presenilin-1 helix-9 "PS1HL9" K V 4.3(70-90) "site-2" and K V 4.3(2-22) "site 1". Namely, dissociation constants for Pb 2+ -bound DREAM interaction with PS1HL9 ( K d = 2.4 ± 0.1 μM), site-2 ( K d = 11.0 ± 0.5 μM) and site 1 ( K d = 5.0 ± 0.6 μM) are nearly identical to those observed for Ca 2+ bound DREAM. Isothermal titration calorimetry data reveal that Pb 2+ binds to two high-affinity sites in Ca 2+ bound DREAM with the overall apparent constant of 4.81 ± 0.06 μM and its binding to Ca 2+ bound DREAM is entropy-driven. Taking into account the structural and sequence similarity between DREAM and other neuronal calcium sensor (NCS) proteins, these results strongly indicate that DREAM and possibly other NCS proteins bind Pb 2+ with a higher affinity than that for Ca 2+ and Pb 2+ interactions with NCS proteins can contribute to Pb 2+ -induced neurotoxicity.

In the present study, we characterize the binding of divalent cations to insulin in aqueous salt solutions by means of capillary electrophoresis and molecular dynamics simulations. The results show a strong pH dependence. At low pH, at which all the carboxylate groups are protonated and the protein has an overall positive charge, all the cations exhibit only weak and rather unspecific interactions with insulin. In contrast, at close to neutral pH, when all the carboxylate groups are deprotonated and negatively charged, the charge-neutralizing effect of magnesium, calcium, and zinc, in particular, on the electrophoretic mobility of insulin is significant. This is also reflected in the results of molecular dynamics simulations showing accumulation of cations at the protein surface, which becomes smaller in magnitude upon effective inclusion of electronic polarization via charge rescaling.

Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions is important in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine, and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

Amylin peptides are secreted together with insulin and zinc ions from pancreatic β-cells. Under unknown conditions, the amylin peptides aggregate to produce oligomers and fibrils, and in some cases Zn 2+ ions can bind to amylin peptides to form Zn 2+ -aggregate complexes. Consequently, these aggregates lead to the death of the β-cells and a decrease in insulin, which is one of the symptoms of type-2 diabetes (T2D). Therefore, it is crucial to investigate the binding sites of the Zn 2+ ions in fibrillary amylin. It was previously found by in vitro and simulation studies that Zn 2+ ion binds to two or four His residues in the turn domain of fibrillary amylin. In the current study, we present a new Zn 2+ binding site in the N-terminus of fibrillary amylin with three different coordination modes. Our simulations showed that Zn 2+ ions bind to polymorphic amylin fibrils with a preference to bind to four Cys residues rather than two Cys residues of two neighboring amylin monomers. The new binding site leads to conformational changes, increases the number of polymorphic states, and demonstrates the existence of competition between various binding sites. Our study provides insight into the molecular mechanisms through which Zn 2+ ions that play a critical role in amylin aggregation can bind to amylin and promote amylin aggregation in T2D.

The mechanism of Aβ aggregation in the absence of metal ions is well established, yet the role that Zn 2+ and Cu 2+ , the two most studied metal ions, released during neurotransmission, paly in promoting Aβ aggregation in the vicinity of neuronal synapses remains elusive. Here we report the kinetics of Zn 2+ binding to Aβ and Zn 2+ /Cu 2+ binding to Aβ-Cu to form ternary complexes under near physiological conditions (nM Aβ, μM metal ions). We find that these reactions are several orders of magnitude slower than Cu 2+ binding to Aβ. Coupled reaction-diffusion simulations of the interactions of synaptically released metal ions with Aβ show that up to a third of Aβ is Cu 2+ -bound under repetitive metal ion release, while any other Aβ-metal complexes (including Aβ-Zn) are insignificant. We therefore conclude that Zn 2+ is unlikely to play an important role in the very early stages (i.e., dimer formation) of Aβ aggregation, contrary to a widely held view in the subject. We propose that targeting the specific interactions between Cu 2+ and Aβ may be a viable option in drug development efforts for early stages of AD.

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, key questions remain about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straightforward interpretation of the measured activity-pH data implies the pK a value of the N1 position in the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pK a shift of G12 toward neutrality. We present computational results that support this hypothesis and provide a model that unifies the interpretation of available structural and biochemical data and paints a detailed mechanistic picture of the general base step of the reaction. Experimentally testable predictions are made for mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Genetically encoded fluorescent Zn(2+) indicators (GEZIs) are highly attractive research tools for studying Zn(2+) homeostasis and signaling in mammalian cells. Most current GEZIs are based on Förster resonance energy transfer (FRET) between a select pair of fluorescent proteins (FPs) fused with Zn(2+)-binding motifs. One drawback of such FRET-based GEZIs is their broad spectral profile bandwidths, creating challenges when monitoring multiple targets or parameters. To address this issue, we have engineered a group of intensiometric GEZIs based on single teal and red FPs that can be utilized to monitor subcellular Zn(2+) diffusion and glucose-induced Zn(2+) secretion in pancreatic INS-1E β-cells. These GEZIs offer the simplicity of intensiometric measurements, compatibility in multicolor imaging, large dynamic ranges, and relatively small molecular sizes, making them valuable additions to the molecular toolbox for imaging Zn(2+).

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.

Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.

An exceptional probe comprising indole-3-carboxaldehyde fluorescein hydrazone (FI) performs multiple tasks, namely, disaggregating amyloid β (Aβ) aggregates in different biomarker environments such as cerebrospinal fluid (CSF), Aβ1-40 fibrils, β-amyloid lysozyme aggregates (LA), and U87 MG human astrocyte cells. Additionally, the probe FI binds with Cu(2+) ions selectively, disrupts the Aβ aggregates that vary from few nanometers to micrometers, and prevents their reaggregation, thereby performing disaggregation and modulation of amyloid-β in the presence as well as absence of Cu(2+) ion. The excellent selectivity of probe FI for Cu(2+) was effectively utilized to modulate the assembly of metal-induced Aβ aggregates by metal chelation with the "turn-on" fluorescence via spirolactam ring opening of FI as well as the metal-free Aβ fibrils by noncovalent interactions. These results confirm that FI has exceptional ability to perform multifaceted tasks such as metal chelation in intracellular conditions using Aβ lysozyme aggregates in cellular environments by the disruption of β-sheet rich Aβ fibrils into disaggregated forms. Subsequently, it was confirmed that FI had the ability to cross the blood-brain barrier and it also modulated the metal induced Aβ fibrils in cellular environments by "turn-on" fluorescence, which are the most vital properties of a probe or a therapeutic agent. Furthermore, the morphology changes were examined by atomic force microscopy (AFM), polarizable optical microscopy (POM), fluorescence microscopy, and dynamic light scattering (DLS) studies. These results provide very valuable clues on the Aβ (CSF Aβ fibrils, Aβ1-40 fibrils, β-amyloid lysozyme aggregates) disaggregation behavior via in vitro studies, which constitute the first insights into intracellular disaggregation of Aβ by "turn-on" method thereby influencing amyloidogenesis.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

Conformational preferences have been surveyed for divalent metal cation complexes with the dipeptide ligands AlaPhe, PheAla, GlyHis, and HisGly. Density functional theory results for a full set of complexes are presented, and previous experimental infrared spectra, supplemented by a number of newly recorded spectra obtained with infrared multiple photon dissociation spectroscopy, provide experimental verification of the preferred conformations in most cases. The overall structural features of these complexes are shown, and attention is given to comparisons involving peptide sequence, nature of the metal ion, and nature of the side-chain anchor. A regular progression is observed as a function of binding strength, whereby the weakly binding metal ions (Ba(2+) to Ca(2+)) transition from carboxylate zwitterion (ZW) binding to charge-solvated (CS) binding, while the stronger binding metal ions (Ca(2+) to Mg(2+) to Ni(2+)) transition from CS binding to metal-ion-backbone binding (Iminol) by direct metal-nitrogen bonds to the deprotonated amide nitrogens. Two new sequence-dependent reversals are found between ZW and CS binding modes, such that Ba(2+) and Ca(2+) prefer ZW binding in the GlyHis case but prefer CS binding in the HisGly case. The overall binding strength for a given metal ion is not strongly dependent on the sequence, but the histidine peptides are significantly more strongly bound (by 50-100 kJ mol(-1)) than the phenylalanine peptides.

Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn(2+), but distinctly different concentrations have been reported when using these sensors to measure Zn(2+) concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn(2+) with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn(2+) in the cytosol but was also successfully used for measuring Zn(2+) in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn(2+) simultaneously in the cytosol and the ER or mitochondria.

Herein, the apparent equilibrium dissociation constant, K(Dapp), between Cu(2+) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), a negatively charged phospholipid, was measured as a function of PS concentrations in supported lipid bilayers (SLBs). The results indicated that K(Dapp) for Cu(2+) binding to PS-containing SLBs was enhanced by a factor of 17,000 from 110 nM to 6.4 pM as the PS density in the membrane was increased from 1.0 to 20 mol %. Although Cu(2+) bound bivalently to POPS at higher PS concentrations, this was not the dominant factor in increasing the binding affinity. Rather, the higher concentration of Cu(2+) within the double layer above the membrane was largely responsible for the tightening. Unlike the binding of other divalent metal ions such as Ca(2+) and Mg(2+) to PS, Cu(2+) binding does not alter the net negative charge on the membrane as the Cu(PS)2 complex forms. As such, the Cu(2+) concentration within the double layer region was greatly amplified relative to its concentration in bulk solution as the PS density was increased. This created a far larger enhancement to the apparent binding affinity than is observed by standard multivalent effects. These findings should help provide an understanding on the extent of Cu(2+)-PS binding in cell membranes, which may be relevant to biological processes such as amyloid-β peptide toxicity and lipid oxidation.

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li(+), Mg(2+), Ca(2+), and Sr(2+); in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na(+), K(+), NH4(+), and Ba(2+). Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg(2+). Additionally, rate-pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg(2+). Results from the thio effect experiments, rate-pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases.

Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²⁺, Zn²⁺, Mn²⁺, or Cd²⁺). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²⁺ concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.

Zinc (Zn2+) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn2+ concentration in the cytosol has been established to be 0.1-1 nM, the free Zn2+ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Förster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondrial matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of ∼300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER [Zn2+] was found to be ≥5 nM, which is >5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn2+ store. Treatment of β-cells or cardiomyocytes with sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors, mobilization of ER Ca2+ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on [Zn2+]ER. These findings question the previously proposed role of Ca2+ in Zn2+ mobilization from the ER and suggest that high ER Zn2+ levels may be an important aspect of cellular homeostasis.

Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.

Metal cations represent key elements of RNA structure and function. In the Neurospora VS ribozyme, metal cations play diverse roles; they are important for substrate recognition, formation of the active site, and shifting the pKa's of two key nucleobases that contribute to the general acid-base mechanism. Recently, we determined the NMR structure of the A730 loop of the VS ribozyme active site (SLVI) that contributes the general acid (A756) in the enzymatic mechanism of the cleavage reaction. Our studies showed that magnesium (Mg(2+)) ions are essential to stabilize the formation of the S-turn motif within the A730 loop that exposes the A756 nucleobase for catalysis. In this article, we extend these NMR investigations by precisely mapping the Mg(2+)-ion binding sites using manganese-induced paramagnetic relaxation enhancement and cadmium-induced chemical-shift perturbation of phosphorothioate RNAs. These experiments identify five Mg(2+)-ion binding sites within SLVI. Four Mg(2+) ions in SLVI are associated with known RNA structural motifs, including the G-U wobble pair and the GNRA tetraloop, and our studies reveal novel insights about Mg(2+) ion binding to these RNA motifs. Interestingly, one Mg(2+) ion is specifically associated with the S-turn motif, confirming its structural role in the folding of the A730 loop. This Mg(2+) ion is likely important for formation of the active site and may play an indirect role in catalysis.

Many proteins use Asx and Glx (x = n, p, or u) side chains as key functional groups in enzymatic catalysis and molecular recognition. In this study, NMR spin relaxation experiments and molecular dynamics simulations are used to measure the dynamics of the side chain amide and carboxyl groups, (13)C(γ/δ), in Escherichia coli ribonuclease HI (RNase H). Model-free analysis shows that the catalytic residues in RNase H are preorganized on ps-ns time scales via a network of electrostatic interactions. However, chemical exchange line broadening shows that these residues display significant conformational dynamics on μs-ms time scales upon binding of Mg(2+) ions. Two groups of catalytic residues exhibit differential line broadening, implicating distinct reorganizational processes upon binding of metal ions. These results support the "mobile metal ion" hypothesis, which was inferred from structural studies of RNase H.

The usually negative, but variable electrical potential (ψ0) at the cell membrane (CM) surface influences the surface activities of free ions and the electrical driving force for the transport of ions across the CM. The rhizotoxic effects and uptake of Zn(2+) and Co(2+) singly and in binary mixture in wheat ( Triticum aestivum L.) at three pH values (4.5, 5.5, or 6.1) were examined in terms of the free ion activities of Zn(2+), Co(2+), and H(+) at the CM surface (these ions are denoted {M(n+)}(0)). Toxicity and uptake of Zn(2+) or Co(2+) singly to roots were better correlated with {M(2+)}(0) than with their bulk-phase activities. Studies of toxicant interactions using the electrostatic approach and a response-multiplication model for toxicant mixtures indicated that {Co(2+)}(0) significantly enhanced the toxicity of {Zn(2+)}(0), but {Zn(2+)}(0) did not significantly affect the toxicity of {Co(2+)}(0). {H(+)}(0) substantially enhanced the toxicity of both metal ions. Taking ψo into account improved the correspondence (denoted r(2)) between observed and predicted uptake of both Zn(2+) and Co(2+), and each inhibited the uptake of the other. Results showed that r(2) increased from 0.776 to 0.936 for Zn uptake and improved from 0.805 to 0.951 for Co uptake. Thus electrostatic models for metal toxicity and uptake proved superior to models incorporating only bulk-phase activities of ions.

The aminoglycoside nucleotidyltransferase (4') (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer-dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg(2+), Mn(2+), and Ca(2+), to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters.

Phosphatidylserine (PS) embedded within supported lipid bilayers was found to bind Cu(2+) from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu(2+)-to-PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85-90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu(2+) concentrations and basic pH values (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion-limited complex formation at basic pH but rapid dissociation under acidic conditions. The tight binding of Cu(2+) to PS may have physiological consequences under certain circumstances.

Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.

Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains., (© 2011 American Chemical Society)

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.

The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu(2+) ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10(-5) to 10(-7) M and increased with increasing activities of H(+), Ca(2+) and Mg(2+) according to the BLM concept. The solution based Cu(2+) EC50 values of N. europaea in six contaminated soils ranged 2 × 10(-6) to 2 × 10(-9) M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu(2+) EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu(2+) in solution represents the toxic species to this bacterium.

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

The CorA Mg(2+) channel is the primary uptake system in about half of all bacteria and archaea. However, the basis for its Mg(2+) selectivity is unknown. Previous data suggested that CorA binds a fully hydrated Mg(2+) ion, unlike other ion channels. The crystal structure of Thermotoga maritima CorA shows a homopentamer with two transmembrane segments per monomer connected by a short periplasmic loop. This highly conserved loop, (281)EFMPELKWS(289) in Salmonella enterica serovar Typhimurium CorA, is the only portion of the channel outside of the cell, suggesting a role in cation selectivity. Mutation of charged residues in the loop, E281 and K287, to any of several amino acids had little effect, demonstrating that despite conservation electrostatic interactions with these residues are not essential. While mutation of the universally conserved E285 gave a minimally functional channel, E285A and E285K mutants were the most functional, again indicating that the negative charge at this position is not a determining factor. Several mutations at K287 and W288 behaved anomalously in a transport assay. Analysis indicated that mutation of K287 and W288 disrupts cooperative interactions between distinct Mg(2+) binding sites. Overall, these results are not compatible with electrostatic interaction of the Mg(2+) ion with the periplasmic loop. Instead, the loop appears to form an initial binding site for hydrated Mg(2+), not for the dehydrated cation. The loop residues may function to accelerate dehydration of the before entry of Mg(2+) into the pore of the channel.